Design, construction, and application of noncanonical redox cofactor infrastructures.

Controlling the flow of carbon and reducing power in biological systems is a central theme in metabolic engineering. Often, trade-offs in pushing carbon flux through targeted pathways while operating in conditions agreeable to the host are required due to the central pools of the shared native redox cofactors NAD(P)/H. Noncanonical redox cofactors (NRCs) have emerged as promising tools to transform how engineers develop biotransformation systems. These new-to-Nature redox cofactors have been demonstrated to function orthogonally to the endogenous cofactors, support pathway thermodynamics optimization, and achieve product scopes previously difficult to reach due to endogenous pathway crosstalk. This review will discuss the development of NRC infrastructures, comprising NRC pools, cofactor reduction sources, and cofactor oxidation sinks, the (pool-source-sink) infrastructure.

Shifting Redox Reaction Equilibria on Demand Using an Orthogonal Redox Cofactor.

Natural metabolism relies on chemical compartmentalization of two redox cofactors, NAD+ and NADP+, to orchestrate life-essential redox reaction directions. However, in whole cells the reliance on these canonical cofactors limits flexible control of redox reaction direction as these reactions are permanently tied to catabolism or anabolism. In cell-free systems, NADP+ is too expensive in large scale. We have previously reported the use of nicotinamide mononucleotide, (NMN+) as a low-cost, noncanonical redox cofactor capable of specific electron delivery to diverse chemistries. Here, we present Nox Ortho, an NMNH-specific water-forming oxidase, that completes the toolkit to modulate NMNH/NMN+ ratio. This work uncovers an enzyme design principle that succeeds in parallel engineering of six butanediol dehydrogenases as NMN(H)-orthogonal biocatalysts consistently with a 103 - 106 -fold cofactor specificity switch from NAD(P)+ to NMN+. We combine these to produce chiral-pure 2,3-butanediol (Bdo) isomers without interference from NAD(H) or NADP(H) in vitro and in E. coli cells. We establish that NMN(H) can be held at a distinct redox ratio on demand, decoupled from both NAD(H) and NADP(H) redox ratios in vitro and in vivo.

Smartphone-Based Paper Microfluidic Particulometry of Norovirus from Environmental Water Samples at the Single Copy Level.

Human enteric viruses can be highly infectious and thus capable of causing disease upon ingestion of low doses ranging from 100 to 102 virions. Norovirus is a good example with a minimum infectious dose as low as a few tens of virions, that is, below femtogram scale. Norovirus detection from commonly implicated environmental matrices (water and food) involves complicated concentration of viruses and/or amplification of the norovirus genome, thus rendering detection approaches not feasible for field applications. In this work, norovirus detection was performed on a microfluidic paper analytic device without using any sample concentration or nucleic acid amplification steps by directly imaging and counting on-paper aggregation of antibody-conjugated, fluorescent submicron particles. An in-house developed smartphone-based fluorescence microscope and an image-processing algorithm isolated the particles aggregated by antibody-antigen binding, leading to an extremely low limit of norovirus detection, as low as 1 genome copy/µL in deionized water and 10 genome copies/µL in reclaimed wastewater.